Methyl-beta-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils

The bioremediation of aged polychlorinated biphenyl (PCB)-contaminated soils is adversely affected by the low bioavailability of the pollutants. Randomly methylated-beta-cyclodextrins (RAMEB) were tested as a potential PCB-bioavailability-enhancing agent in the aerobic treatment of two aged-contaminated soils. The soils, contaminated by about 890 and 8500 mg/kg of Aroclor 1260 PCBs, were amended with biphenyl (4 g/kg), inorganic nutrients (to adjust their C:N ratio to 20:1), and variable amounts of RAMEB (0%, 0.5%, or 1.0% [w/w]) and treated in both aerobic 3-L solid-phase reactors and 1.5-L packed-bed loop reactors for 6 months. Notably, significant enhancement of the PCB biodegradation and dechlorination, along with a detectable depletion of the initial soil ecotoxicity, were generally observed in the RAMEB-treated reactors of both soils. RAMEB effects were different in the two soils, depending upon the treatment conditions employed, and generally increased proportionally with the concentration at which RAMEB was applied. RAMEB, which was slowly metabolized by the soil's aerobic microorganisms, was found to markedly enhance the occurrence of the indigenous aerobic, cultivable biphenyl-growing bacteria harboring genes homologous to those of two highly specialized PCB degraders (i.e., bphABC genes of Pseudomonas pseudoalcaligenes KF707 and bphA1A2A3A4BC1 genes of Rhodococcus globerulus P6) and chlorobenzoic acid-degrading bacteria as well as the occurrence of PCBs in the water phase of the soil reactors. These findings indicate that RAMEB enhanced the aerobic bioremediation of the two soils by increasing the bioavailability of PCBs and the occurrence of specialized bacteria in the soil reactors.     

An O Antigen Capsule Modulates Bacterial Pathogenesis in Shigella sonnei

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.     

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.     

Aerobic cometabolism of chloroform by butane-grown microorganisms: long-term monitoring of depletion rates and isolation of a high-performing strain

The focus of this microcosm study was to monitor the performances of 17 butane-utilizing microcosms during a long-term (100–250 days) aerobic cometabolic depletion of chloroform (CF). The depletion of the contaminant began after a lag-time variable between 0 and 23 days. All microcosms quickly reached a pseudo steady-state condition, in terms of biomass concentration (with an average of 9.3 × 10⁶ CFU ml^–1), chloroform depletion rate (5 mol l^–1 d^–1) and butane utilization rate (730 mol l^–1 d^–1). After about 100 days of CF depletion, a sudden 5- to 7-fold increase of the chloroform rate was observed in two microcosms, where the highest amount of contaminant had been depleted. In one of these high-performing microcosms, an experiment of chloroform depletion in the absence of butane resulted in the depletion of a surprisingly high amount of contaminant (765 mol_CF kg_{dry soil}^–1 in 2 months) and in a marked selection of a single bacterial strain. Bioaugmentation assays conducted with the biomass selected in this microcosm and with a pure culture of the selected strain immediately resulted in very high chloroform depletion rates. Preliminary results of a study conducted with resting cells of the selected strain indicated that it can degrade chloroform concentrations up to 119 M (14.2 mg l^–1) without any sign of substrate toxicity, and that it is able to transform vinyl chloride and 1,1,2-trichloroethane.     

Epidermal growth factor and betacellulin mediate signal transduction through co-expressed ErbB2 and ErbB3 receptors.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.     

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.     

Molecular mechanism underlying impaired platelet responsiveness in liver cirrhosis

We have studied platelet function in 10 patients with severe liver cirrhosis, compared to healthy subjects. Using washed platelets, we have investigated the molecular mechanism underlying the defect in platelet aggregation frequently observed in these patients. We have found that platelets from cirrhotic patients have a reduced responsiveness to thrombin and collagen in terms of aggregation, and receptor-dependent activation of phospholipase C, A2 and cyclooxygenase/thromboxane synthetase. We thus suggest that this impairment in transmembrane signalling is responsible for the defective platelet function observed in cirrhosis.     

Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells

Ultraviolet A (UVA) radiation represents more than 90% of the UV spectrum reaching Earth's surface. Exposure to UV light, especially the UVA part, induces the formation of photoexcited states of cellular photosensitizers with subsequent generation of reactive oxygen species (ROS) leading to damages to membrane lipids, proteins and nucleic acids. Although UVA, unlike UVC and UVB, is poorly absorbed by DNA, it inhibits cell cycle progression, especially during S-phase. In the present study, we examined the role of the DNA damage checkpoint response in UVA-induced inhibition of DNA replication. We provide evidence that UVA delays S-phase in a dose dependent manner and that UVA-irradiated S-phase cells accumulate in G2/M. We show that upon UVA irradiation ATM-, ATR- and p38-dependent signalling pathways are activated, and that Chk1 phosphorylation is ATR/Hus1 dependent while Chk2 phosphorylation is ATM dependent. To assess for a role of these pathways in UVA-induced inhibition of DNA replication, we investigated (i) cell cycle progression of BrdU labelled S-phase cells by flow cytometry and (ii) incorporation of [methyl-(3)H]thymidine, as a marker of DNA replication, in ATM, ATR and p38 proficient and deficient cells. We demonstrate that none of these pathways is required to delay DNA replication in response to UVA, thus ruling out a role of the canonical S-phase checkpoint response in this process. On the contrary, scavenging of UVA-induced reactive oxygen species (ROS) by the antioxidant N-acetyl-l-cystein or depletion of vitamins during UVA exposure significantly restores DNA synthesis. We propose that inhibition of DNA replication is due to impaired replication fork progression, rather as a consequence of UVA-induced oxidative damage to protein than to DNA.